Methods and kits for extraction of DNA

ABSTRACT

Methods and materials are disclosed for use in recovering a biopolymer from a solution. In particular, the invention provides methods for extraction and isolation of nucleic acids from biological materials. The nucleic acids can be separated by forming a stable complex with soluble polysaccharide polymers and magnetic particles, in the presence of detergents and solvent. When the particles are magnetically separated out of the solution, the nucleic acids are separated with them. The nucleic acids can subsequently be released and separated from the particles. The nucleic acid preparation is useful for achieving efficient and accurate results in downstream molecular techniques such as quantification, identification of the source of the nucleic acids, and genotyping.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.15/216,642 filed Jul. 21, 2016, which is a divisional of U.S.application Ser. No. 13/931,110 filed Jun. 28, 2013 (now U.S. Pat. No.9,422,544), which is a continuation of U.S. application Ser. No.12/371,515 filed Feb. 13, 2009 (now U.S. Pat. No. 8,497,065), whichclaims the benefit of priority under 35 U.S.C. 119 to U.S. ProvisionalApplication No. 61/028,986 filed Feb. 15, 2008, all of which areincorporated herein by reference in their entireties.

INTRODUCTION

The present teachings are generally directed to methods of isolatingnucleic acids from biological materials, such that the nucleic acids arecompatible with use in downstream applications. In various embodiments,the present teachings relate to the use of polyhydroxy polymers anddetergents for binding of nucleic acids to, and release of nucleic acidsfrom, magnetic particles with hydrophilic surfaces. In some embodiments,kits are provided for isolating DNA from biological materials.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way. All literature and similar materials cited in this application,including but not limited to patents, patent applications, articles,books and treatises, regardless of the format of such literature andsimilar materials, are expressly incorporated by reference in theirentirety for any purpose.

DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 is a schematic demonstrating a method of DNA isolation andpurification, as described in various embodiments of the presentteachings.

FIG. 2 demonstrates DNA yield from DNA isolation and purification asdescribed in Example 6, wherein genomic DNA was isolated from culturedRaji cells at cell counts varying from 1562 to 50000, according to themethods described in Example 4.

FIG. 3 demonstrates DNA yield from DNA isolation and purification asdescribed in Example 7, wherein genomic DNA was isolated from culturedK562 cells at cell counts varying from 3500 to 110000, according to themethods as described in the Example 4.

FIGS. 4A-4E demonstrate genotyping profiles obtained following DNAisolation and purification from substrates as described in Example 13,wherein genomic DNA was isolated from biological samples according tothe methods of Example 11, and was processed for genotyping using theIdentifiler® kit.

DESCRIPTION OF VARIOUS EMBODIMENTS

While the present teachings are described in conjunction with variousembodiments, it is not intended that the present teachings be limited tosuch embodiments. On the contrary, the present teachings encompassvarious alternatives, modifications and equivalents, as will beappreciated by those of skill in the art.

Most of the words used in this specification have the meaning that wouldbe attributed to those words by one skilled in the art. Wordsspecifically defined in the specification have the meaning provided inthe context of the present teachings as a whole, and as are typicallyunderstood by those skilled in the art. In the event that a conflictarises between an art-understood definition of a word or phrase and adefinition of the word or phrase as specifically taught in thisspecification, the specification shall control. Headings used herein aremerely for convenience, and are not to be construed as limiting in anyway.

As used herein, “DNA” refers to deoxyribonucleic acid in its variousforms as understood in the art, such as genomic DNA, cDNA, isolatednucleic acid molecules, vector DNA, and chromosomal DNA. “Nucleic acid”refers to the nucleic acid molecule or molecules, DNA or RNA(ribonucleic acid) in any form. As used herein, the term “isolatednucleic acid molecule” or “isolated nucleic acid” refers to a nucleicacid molecule (DNA or RNA of any form) that has been removed from itsnative environment. Some examples of isolated nucleic acid molecules arerecombinant DNA molecules contained in a vector, recombinant DNAmolecules maintained in a heterologous host cell, partially orsubstantially purified nucleic acid molecules, nucleic acids obtainedfrom forensic and other samples comprising biological material, such asblood, semen, saliva, skin tissue, etc., and synthetic DNA molecules. An“isolated” nucleic acid can be free of sequences which naturally flankthe nucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. Moreover, an “isolated” nucleic acid molecule, such asa cDNA molecule, can be substantially free of other cellular material orculture medium when produced by recombinant techniques, or of chemicalprecursors or other chemicals when chemically synthesized.

“Polymerase chain reaction” (or “PCR”) refers to a technique in whichrepetitive cycles of denaturation, annealing with a primer, andextension with a DNA polymerase enzyme are used to amplify the number ofcopies of a target DNA sequence by approximately 10⁶ times or more. ThePCR process for amplifying nucleic acids is covered by U.S. Pat. Nos.4,683,195 and 4,683,202, which are herein incorporated in their entiretyby reference for a description of the process. The reaction conditionsfor any PCR comprise the chemical components of the reaction and theirconcentrations, the temperatures used in the reaction cycles, the numberof cycles of the reaction, and the durations of the stages of thereaction cycles.

“PCR-compatible” refers to any composition, solution, compound, reagent,etc. that is compatible with subsequent use in PCR assays and isrelatively non-inhibitory of the enzymatic polymerase chain reaction.PCR-compatible products demonstrate relatively minimal or no inhibitionof PCR amplification, as evidenced by comparison of PCR results with therelevant positive and negative controls. PCR assays can include, but arenot limited to, DNA genotyping systems, TaqMan® or SYBR® green real-timePCR assays for DNA quantification, multiplex PCR assays including thosedesigned to genotype short tandem repeats, etc.

As used herein, “amplify” refers to the process of enzymaticallyincreasing the amount of a specific nucleotide sequence. Thisamplification is not limited to but is generally accomplished by PCR.

“Polymer” or “polymers” refer to soluble polyhydroxy polymers forbinding of nucleic acids to, and release of nucleic acids from, magneticparticles with hydrophilic surfaces.

In some embodiments of the present teachings, methods are described inwhich nucleic acid molecules can be separated and/or isolated fromsamples and, in some embodiments, in which the product made from themethods are nucleic acids. In some embodiments, the methods of thepresent teachings result in the formation of a product which comprisesthe isolated nucleic acid.

In some embodiments of the present teachings, nucleic acid molecules canbe separated and/or isolated from samples containing biologicalmaterials and, in some embodiments, the product made from the methodsare nucleic acids. Examples of such samples include but are not limitedto blood and blood stains, saliva and saliva stains, buccal cells andbuccal swabs, semen and semen stains, cigarette butts, and chewing gum.

In some embodiments of the present teachings, methods are describedwherein a sample can be treated with a starting solution comprisingsoluble polyhydroxy polymers and detergent, and adding magneticallyattractable particles in order to recover nucleic acid molecules fromthe sample and, in some embodiments, nucleic acids are recovered fromthe sample by applying a magnetic field. In various embodiments, thesample can comprise one or more of free nucleic acids; cells; biologicalmaterials such as buccal swabs, stained fabrics, and so on. Inadditional embodiments of the present teachings, methods are describedwherein a nucleic acid can be separated from a sample, comprising thesteps of treating the sample with a starting solution comprising apolymer and detergent; adding suspended magnetically attractableparticles to the treated sample; and separating the nucleic acidattached to the magnetically attractable particles via the polymer byapplying a magnetic field. Such methods may further comprise the step ofreleasing the nucleic acid from the magnetically attractable particles.Such methods may yet further comprise the step of eluting the nucleicacid in an aqueous solution.

The nucleic acids thus obtained can then be utilized in any of variousdownstream applications such as, for example, quantification, detection,and genotyping of specific nucleic acids or even of a biologicalspecies. These analyses can be performed, for example, by PCRamplification. As one example, in forensic DNA analysis the humannuclear DNA (nDNA) and/or genomic DNA can be obtained from complexbiomaterials and then genotyped using PCR. As another example, a DNApreparation can be used for quantification of human DNA, or morespecifically human male DNA, using real-time PCR systems such asQuantifiler®, and/or genotyped for autosomal or Y-chromosomal shorttandem repeat loci using systems such as, for example, AmpFISTR® kits.Based upon the amount of DNA present in a sample, a particulargenotyping system can be selected that will yield the best results forthe particular analysis required. Therefore, in order to best utilizenucleic acids in downstream applications, it is particularly desirablethat the methods result not only in a product of high yield, but alsoone that is relatively free of inhibitors of downstream applicationssuch as PCR.

As an example, typical forensic evidence samples are often exposed tovarious environmental insults during acquisition and processing, whichcan lead to contamination with compounds that act to inhibit PCR, andwhich therefore interfere with attempts at genotyping or other analyses.It is desirable to remove such inhibitors during the isolation of DNAand prior to amplification.

Various embodiments of the present teachings relate to efficient bindingof nucleic acids such as, for example, genomic DNA to magnetic particles(i.e., magnetically attractable particles, or beads) in such a form thatthe bound nucleic acids can then be released under the appropriateaqueous conditions. Embodiments of these teachings thus enable effectiveisolation of nucleic acids, such as genomic DNA, from various differenttypes of biological materials. In addition, nucleic acids such asgenomic DNA can be isolated from either small or large quantities of thebiological materials that are commonly processed in laboratories suchas, for example, those involved in genotyping analyses.

These embodiments and other features of the present teachings willbecome more apparent from the description herein.

Nucleic Acid Isolation System

Various embodiments of the present teachings relate to a system,amenable to assembly in a kit, for the binding of nucleic acids such asgenomic DNA to magnetic particles having a hydrophilic surface viasoluble polyhydroxy polymers, in the presence of detergents, forming anucleic acid-polymer-particle complex, and the production and isolationof such a complex, wherein the nucleic acid does not directly bind tothe magnetic particles. The formation of the complex is such that thepolymer entraps the nucleic acid, polymer attaches to the particles, andso indirectly connects the nucleic acid with the particles. Variousembodiments comprise a lysis solution, which causes the lysis of cellsand release of nucleic acid, while protecting the nucleic acid fromdegradation and removing PCR inhibitors. In various embodiments of thepresent teachings the nucleic acids remain bound to the magneticparticles via the complex in the presence of a wash solution, in whichthe complex is washed so as to remove contaminants and so that thenucleic acid is amendable to use in downstream applications, such asPCR. Solutions for washing nucleic acid isolations of any contaminantsand inhibitors are well-known to those of skill in the art, and cancomprise, for example, detergents and polar solvents. In embodiments ofthe present teachings, the nucleic acids can then be released in anaqueous solution, such as a buffer, which is also compatible for use indownstream applications such as PCR. A plurality of washes can beperformed in the methods of the present invention, separately or inconjunction with a plurality of applications of the magnetic field tothe sample to recover and/or separate the nucleic acids.

Standard nucleic acid extraction techniques, including cell lysis, andwashing and elution of nucleic acids, are well known in the art andunless otherwise noted, can be carried out according to varioustechniques as described, for example, in DNA Typing Protocols: MolecularBiology and Forensic Analysis, 1^(st) edition, B. Budowle et al., eds.,Eaton Publishing Co. (2000); J M Butler, Forensic DNA Typing: Biology,Technology, and Genetics of STR Markers, 2^(nd) edition, ElsevierAcademic Press (2005); or P. Gill, “Application of Low Copy Number DNAProfiling,” Croatian Medical Journal vol. 42, pages 229-232 (2001); F RBieber et al., “Isolation of DNA from Forensic Evidence,” CurrentProtocols in Human Genetics, Supplement 26 (2000); Forensic DNAProfiling Protocols, Methods in Molecular Biology, vol. 98, PJ Lincolnand J. Thomson, eds., Humana Press (1998).

Various embodiments of the present teachings relate to a nucleic acidisolation system, such as for genomic DNA, comprising reagents andmethods for extraction of the nucleic acids from biological samples.Embodiments of these methods can comprise: forming a non-covalentcomplex of nucleic acid (e.g., genomic DNA) with soluble polymers havingthe same or similar chemical structure as the surface of magneticallyattractable particles, in the presence of detergents; binding of thenucleic acid-polymer complex to magnetic particles via interactionsbetween the polymers and surfaces of the particles, thus forming astable nucleic acid-polymer-particle complex; releasing the nucleic acidfrom the particles, and eluting the nucleic acid in an aqueous solution.

Applicants have found that the use of polyhydroxy water-soluble polymersand detergent, in the presence of appropriate salts and polar solvent,improves the efficiency of the binding and release of nucleic acids suchas genomic DNA on the surface of magnetically attractable particles. Anexample of appropriate magnetically attractable particles is, but is notlimited to, dextran-encased Nanomag® magnetite nanoparticles. In someembodiments of the present teachings, dextran-encased magnetitenanoparticles are added to a sample comprising nucleic acids in therange of about 2 to about 10 mg/ml.

The soluble polymers and detergent may be termed binding enhancers. Someexamples of appropriate polyhydroxy water-soluble polymers are, but arenot limited to, long-chain branched polysaccharides such as dextrans(e.g., dextran 5,000,000-40,000,000), soluble starch, dextrins,cellodextrins, polyethylene glycol (PEG), heparin, glycogen, short-chaincellulose, cellulose derivatives, and combinations thereof. Someexamples of appropriate detergents are, but are not limited to,N-lauroyl sarcosine (NLS); lauroyl sarcosinate, also known as sarcosyl,an ionic surfactant derived from sarcosine; hexadecyltrimethylammoniumbromide or cetyltrimethylammonium bromide (CTAB); deoxycholate; dodecylβ-D-maltoside; nonanoyl-N-methylglucamide; sodium dodecyl sulfate;polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether(commercially known as Triton® X-100); and combinations thereof. In someembodiments, the binding enhancer comprises dextran in the range of 1-5mg/ml and sarcosyl in the range of about 5 to about 15%.

Some examples of appropriate polar solvents are, but are not limited to,isopropanol, ethanol, butanol, and combinations thereof. In someembodiments, the polar solvent comprises about 80% to about 100%isopropanol.

In some embodiments, magnetically attractable particles can be added toa sample comprising nucleic acid, such as a cell lysate, at the sametime as the binding enhancers, forming a suspension. A solutioncomprising polar solvent can then be added to this suspension.Alternatively, in some embodiments of the present teachings a singlesolution comprising binding enhancers and polar solvent can be added tothe suspension. Binding enhancers, solvent and cell lysate provideunique conditions such that nucleic acids are entrapped in anon-covalent complex with soluble polymers having the same or a similarchemical structure as the surface of the magnetic particles. The resultis the effective binding of the polymer-entrapped nucleic acids to themagnetic particles in a non-covalent nucleic acid-polymer-particlecomplex. This complex is stable under alcohol wash conditions, and thenucleic acids can easily be later eluted in a standard low-salt buffersuch as, for example, Tris buffer containing low concentrations ofdivalent metal ion chelating agents, such as EDTA. In some embodiments,this stage of nucleic acid-polymer binding to particles may be assistedby chilling.

Following binding of the polymer-entrapped nucleic acids to magneticparticles in the formation of the nucleic acid-polymer-particlecomplexes, a magnetic field can then be applied to the suspension. Thismagnetic field can be used to remove the nucleic acid-polymer-particlecomplexes from the suspension, forming a complex layer, for example, atthe bottom or side of the tube and leaving a supernatant. The magneticfield can be applied to the sample by devices and methods known in theart (e.g., via Ambion® (Austin, Tex.) Magnetic Stands). The supernatantcan then be removed from the tube.

The nucleic acid-polymer-particle complex layer can then optionally bewashed, to remove residual contaminants and/or inhibitors of PCR; thenthe nucleic-acid-polymer-particle complex can be resuspended in therequisite volume of an appropriate elution buffer in the absence of anymagnetic field. The appropriate elution buffers for the isolation ofnucleic acids are well-known to those of skill in the art. These allowfor release of the nucleic acids into solution from the nucleicacid-polymer-particle complexes. A magnetic field can be reapplied tothe tube, resulting in removal of the magnetic particles from thesuspension to, for example, the bottom or side of the tube, leaving asupernatant in which the nucleic acid is now dissolved. The redissolvednucleic acid can now be separated from the magnetic particles bycollecting the supernatant with, for example, a pipette while theparticles are held against the bottom or side of the tube by themagnetic field. Centrifugation of the sample is not required in thesemethods.

In some embodiments of the present teachings, then, nucleic acidmolecules can be isolated from samples containing biological materialsand purified from solution in combination with the use of magneticparticles with a hydrophilic surface, such as, for example, magneticdextran particles. Magnetic particle-facilitated nucleic acid isolationand purification can be used to greatly improve upon the traditionalprocess of alcohol-based precipitation isolation and purification ofnucleic acids, well-known to those of skill in the art. An example of anembodiment of an alcohol-based isolation and purification procedure asmodified by these teachings can be demonstrated by reference to FIG. 1.

Lysis Solution

Embodiments of the methods described herein can comprise the lysing ofcells from biological materials present on a substrate such as, forexample, the cotton of a buccal swab or a stained fabric, to create alysate comprising nucleic acids; removing the substrate from the lysate;forming the nucleic acid-polymer-particle complex, followed byseparating and eluting of nucleic acids as described above. In oneembodiment, the lysis solution can comprise SDS, Tris buffer and NaCl,optionally with proteinase K; and in some embodiments the lysis solutioncan comprise 0.0%4% SDS, 100 mM Tris buffer, and 2 M NaCl.

In various embodiments of the present teachings, a lysis solution can beadded to a sample containing biological material (and optionally, thesample subjected to heat for some time, e.g., twenty minutes to twohours) in order to lyse cells and free nucleic acids into a lysate. Insome embodiments the lysis solution can be a composition comprisingcompounds designed to effectively lyse cells, e.g., buccal cellscollected on a cotton swab, while also protecting released nucleic acidsfrom degradation. In some embodiments the lysis solution furthercomprises such compounds as to ensure that released nucleic acids arecompatible with use in downstream assays such as, for example, PCRassays, and in particular DNA genotyping systems.

In some embodiments of the present teachings, magnetic particles,binding enhancers and polar solvent can then be added to the lysatecomprising the nucleic acid following the lysis procedure, creating asuspension in which the nucleic acid-polymer-particle complexes areformed, and the nucleic acids are separated and eluted as describeabove.

Wash Solution

Various embodiments of the present teachings relate to a nucleic acidisolation system, such as for genomic DNA, comprising reagents andmethods for extraction of the nucleic acids from a biological sample,and comprise a wash step to remove PCR inhibitors from the sample. Thewash solution used in the wash step is such as are known in the art. Insome embodiments, a particular component of the wash solution can beethanol in a concentration ranging from 70%-90%. Embodiments of thesemethods can comprise: formation of a non-covalent complex of nucleicacid (e.g., genomic DNA) with soluble polymers having the same orsimilar chemical structure as the surface of magnetic particles; bindingof the nucleic acid-polymer complex to magnetic particles viainteractions between the polymers and surfaces of the particles, thusforming a stable nucleic acid-polymer-particle complex; removal ofunbound materials, such as PCR inhibitors, via a wash solution reagentcomprising detergent and polar solvent; and elution of the nucleic acidin an aqueous solution amenable to use in downstream applications suchas PCR.

Using a wash solution of detergents and polar solvent to wash thenucleic acid-polymer-particle complex helps remove any residual salt,nucleotides, chemicals, organic solvents, and other contaminants in thesample, and improves the removal of inhibitors of downstreamapplications, such as PCR inhibitors. In various embodiments, a washsolution can be used as a wash of nucleic acid-polymer-particlecomplexes to remove PCR inhibitors and/or contaminants. Solutions usefulfor washing nucleic acids during isolation and/or purification arewell-known to those of skill in the art.

In some embodiments, a wash solution is added to the nucleicacid-polymer-particle complex to create a wash suspension. The nucleicacid is insoluble in alcohol, and remains in a stable complex with thepolymers and particles during washing. The washing step can thus beperformed vigorously (e.g., by vortex mixing) without risk of loss ofthe nucleic acids. The sample can then be placed before a magnetic fieldagain, and the resultant wash supernatant comprising contaminants can beremoved from the complex layer, which separates out of the washsuspension.

After the washing step, if performed, a second wash step can also beperformed following similar steps as in the first wash. In someembodiments of the present teachings, following the wash step(s) thenucleic acids are separated and eluted as describe above.

Nucleic Acid Extraction and Purification

The extraction and purification methods of the present teachings provideuseful methods for obtaining nucleic acids such as genomic DNA frombiological samples which can be used in downstream applications such asgenotyping, quantification, and identification of the source of thebiological material, wherein molecular biological processes such as PCRare utilized. The exemplary results described in the Examples hereinillustrate the various advantages of the nucleic acid extraction andpurification methods of the present teachings, which include but are notlimited to providing a nucleic acid (e.g., genomic DNA) preparation that(a) can be derived from a variety of biological materials, (b) is freeof detectable inhibitors of downstream applications, such as PCRamplification; (c) can be in concentrated form, and, (d) is amenable foruse in any of various applications for nucleic acid analysis, such asgenotyping, quantification, detection of the source of biologicalmaterial, etc. Furthermore, the procedure for extraction andpurification of nucleic acids is fully automatable, using standardliquid handling systems.

Additionally, modification of standard alcohol precipitation procedurescurrently in use, such as those requiring centrifugation, by the methodsof the present teachings can provide several clear benefits. First, themethods herein exemplified by the present teachings are faster—themodified procedure for removal of solution from the separated nucleicacid complex takes only about 1-2 minutes, as opposed to about 10-30minutes for conventional methods using centrifugation. Second, themethods are not reliant upon centrifugation equipment, but can beperformed with a simple magnet setup. Third, the methods are morereadily suited to automation. For example, a great many tubes could beplaced over a large electromagnet and nucleic acids from these could allbe isolated simultaneously using, e.g., a multi-channel pipettingdevice. Fourth, the methods of present teachings are especiallyeffective for step of washing the nucleic acid-polymer-magnetic particlecomplexes (e.g., in order to remove any residual salt, nucleotides ororganic solvents such as phenol), because in the present teachings thewashing steps do not require centrifugation and so can be performedrapidly. Additionally, there is minimal or no risk of loss of material,as can occur with the conventional methods based upon centrifugation,where the pellet often detaches from the bottom of the tube during suchwashing.

As those skilled in the art will appreciate, numerous changes andmodifications may be made to the various embodiments of the presentteachings without departing from the spirit of these teachings. It isintended that all such variations fall within the scope of theseteachings.

All of the compositions and methods of the present teachings, asdisclosed and claimed herein, can be made and executed without undueexperimentation in light of the present disclosure. While thecompositions and methods of these teachings have been described in termsof specific embodiments, it will be apparent to those of skill in theart that variations may be applied to the compositions and methods, andin the steps or in the sequence of steps of the methods describedherein, without departing from the concept and scope of these teachings.More specifically, it will be apparent that certain agents which areboth chemically and physiologically related may be substituted for theagents described herein, while the same or similar results would beachieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the scope of theinvention as defined by the appended claims.

EXAMPLES

Aspects of the present teachings may be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way.

Example 1

Samples of human body fluids were obtained in polypropylene tubes of 2.0ml capacity. The samples were 2 μl blood, 10 μl saliva, and 2 μl semen.Each was mixed with a lysis solution in order to lyse cells. The lysissolution comprised 0.0%4% SDS, 100 mM Tris buffer, and 2 M NaCl,optionally with proteinase K. The lysis mixtures were incubated with orwithout shaking at a temperature in the range of approximately 60° to80° C. for a period of 40 minutes to 1 hour.

The genomic DNA released from the biological materials was then bound tothe magnetic particles having the polyhydroxy groups of dextran. Thebinding mixture of each sample contained the cell lysate, 10 to 20 μl ofa suspension comprising the magnetic particles at a concentration ofapproximately 5-20 mg/ml, and 150 to 300 μl of a polar compound suchisopropanol, ethanol, and/or PEG. The DNA bound to the magneticparticles was then physically separated from the binding mixture by theapplication of a magnetic field to the mixture.

The DNA, still bound to the magnetic particles in a complex, was thenwashed with an alcohol-based wash solution (90% Ethanol). TheDNA-magnetic particle complexes were again physically separated from thewash mixture by use of a magnetic field. The wash step was repeated oneto two times. PCR inhibitors and other macromolecules entrapped in theDNA-magnetic particle complexes were removed in this wash step. The DNAwas then released from the magnetic particles by suspending theDNA-particle complexes in 10 to 100 μl of aqueous solution, such asDNA-free water or a neutral buffer such as Tris-HCl, and this DNArelease mixture was incubated at a temperature in the range ofapproximately 50 to 75° C. Released genomic DNA was then physicallyseparated from the magnetic particles by use of a magnetic field. Thegenomic DNA preparation thus obtained was stored at 4° C. for short-termstorage, or at −20° C. for long-term storage. The DNA was quantified bythe use of standard methods well-known to those of skill in the art. Theresults thus obtained, typical for human genomic DNA, are presented inTable 1.

TABLE 1 Sample Yield of DNA, ng 2 μl liquid blood 8 2 μl liquid semen250 10 μl liquid saliva 100

Example 2

Genomic DNA from biological samples was extracted and isolated asdescribed in Example 1, wherein the binding mixture comprised: the celllysate; 10 to 20 μl of the magnetic particle suspension wherein theparticles possessed the polyhydroxy groups of dextrans; 10 to 20 μl ofpolyhydroxy polysaccharides such as dextran, cellulose, or solublestarch, at concentrations ranging from approximately 1 to 10 mg/ml; 10to 20 μl of anionic detergents, such as N-lauroyl sarcosine, sodiumdeoxycholate, CTAB, N-dodecyl β-D-maltoside, nonanoyl-N-methylglucamide,Triton® X-100 or sodium dodecyl sulfate; and 150 to 300 μl of a polarcompound such as isopropanol, ethanol, and/or PEG. The yield of genomicDNA obtained from this method is presented in Table 2.

TABLE 2 Sample Yield of DNA, ng 2 μl liquid blood 140 2 μl liquid semen1200 10 μl liquid saliva 200

Example 3

Genomic DNA from biological samples was extracted and isolated asdescribed in Example 2, wherein the inhibitors and other macromoleculesentrapped in the DNA bound to the magnetic particles were removed usingtwo different wash solutions. The step of washing the DNA bound to themagnetic particles comprised one wash with the first wash solution, thenone to two wash steps using the second wash solution.

Example 4

Samples of the human body fluids 2 μl of blood, 20 μl of saliva, and 1μl of semen were obtained in a polypropylene tube of 2.0 ml capacity andmixed with a lysis solution. The lysis mixture was incubated with orwithout shaking at a temperature in the range of approximately 60 to 80°C. for a period of approximately 40 minutes to 4 hours.

The genomic DNA released from the biological materials was then bound tothe magnetic particles comprising the polyhydroxy groups of dextran. Thebinding mixture comprised: the cell lysate; 10 to 20 μl of thesuspension comprising the magnetic particles at a concentration in therange of approximately 5-20 mg/ml; 10 to 20 μl of polyhydroxypolysaccharides, such as dextran, cellulose, and/or soluble starch at aconcentration in the range of approximately 1 to 10 mg/ml; approximately10 to 20 μl of anionic detergents, such as N-lauroyl sarcosine, sodiumdeoxycholate, CTAB, N-dodecyl β-D-maltoside, nonanoyl-N-methylglucamide,Triton® X-100, and/or sodium dodecyl sulfate; and approximately 150 to300 μl of a polar compound such as isopropanol, ethanol, and/or PEG.

The DNA bound to the magnetic particles was physically separated fromthe binding mixture by the use of a magnetic field. The DNA bound to themagnetic particles was then washed with a wash solution. The DNA boundto the magnetic particles was physically separated from the wash mixtureby use of a magnetic field. The wash step was repeated one to two times.The PCR inhibitors and other macromolecules entrapped in the DNA boundto the magnetic particles were removed in this step.

The DNA was then released from the magnetic particles by suspending theDNA bound to the magnetic particles in approximately 10 to 100 μl ofaqueous solution, such as DNA-free water; or buffer comprising Tris-HCland the chelating agent. The release mixture was incubated at atemperature in the range of approximately 50 to 75° C. Released genomicDNA was then physically separated from the magnetic particles by use ofa magnetic field. The genomic DNA preparation thus obtained was storedat 4° C. for short-term storage, or at −20° C. for long-term storage.The DNA was quantified using standard methods well-known to those ofskill in the art such as, for example, quantification of human DNA usingthe Quantifiler® Human DNA Quantification Kit. Results thus obtained,typical for human genomic DNA, are presented in Table 3.

TABLE 3 Sample Yield of DNA, ng 2 μl liquid blood 145 1 μl liquid semen650 20 μl liquid saliva 165

Example 5

Genomic DNA was isolated as described in Example 4, wherein thebiological fluids were 2 μl of blood, 20 μl of saliva and 1 μl of semen,and the fluids were stained on fabric such as cotton cloth, polyestercloth or denim. Results thus obtained for fluids stained on cotton,typical for human genomic DNA, are presented in Table 4.

TABLE 4 Sample Yield of DNA, ng 2 μl blood on cotton 80 1 μl semen oncotton 550 20 μl saliva on cotton 145

Example 6

Genomic DNA was isolated from cultured Raji cells at cell counts rangingfrom 1562 to 50000 cells, as described in Example 4. Results thusobtained, typical for human genomic DNA, are presented in FIG. 2 andTable 5.

TABLE 5 Raji Cells, cell count DNA yield, ng 50000 488 25000 266 12500136 6250 69.7 3125 37.3 1562 21

Example 7

Genomic DNA was isolated from cultured K562 cells at cell counts rangingfrom approximately 3500 to 110000 cells, as described in Example 4.Results thus obtained, typical for human genomic DNA, are presented inFIG. 3 and Table 6.

TABLE 6 K562 Cells, cell count DNA yield, ng 110000 1190 55000 700 27500500 13750 217 6875 114 3438 65

Example 8

Genomic DNA was isolated from biological fluids and stains of biologicalfluids on different substrates, such as cloth, FTA paper, a swab anddenim, as described in Example 4, wherein the lysis of biologicalmaterial was performed by suspending the biological material in a lysissolution. The lysis solution was incubated at a temperature in the rangeof 50 to 60° C. for a period of 40 minutes to 2 hours, and DNA wasobtained and quantified by methods as outlined in Example 4.

Example 9

Genomic DNA was isolated as described in Example 4, whereinapproximately 2 to 10 μl of the biological fluid blood was spotted oncotton, and was enriched with 1 to 5 μl of PCR inhibitors comprisinghematin to a final concentration ranging from approximately 0.1 to 2 mM,humic acid to a final concentration ranging from approximately to 5mg/ml, indigo to a final concentration ranging from 5 to 20 mM, andurban dust extract to a final concentration ranging from approximately 2to 12 mg/ml. The PCR inhibitors were effectively removed during theextraction and isolation of the genomic DNA by these methods, asevidenced by measuring the C_(t) value of the internal PCR control (IPC)using the Quantifiler® Human DNA Quantification Kit. Results thusobtained are presented in Table 7.

TABLE 7 Sample IPC C_(t) 2 μl blood dried on cotton 27.58 2 μl blooddried on cotton in presence of inhibitors mix 27.60 Extraction Blank27.77

Example 10

Genomic DNA was isolated as described in the Example 4, wherein thebiological samples comprising stains of biological fluids underwentenvironmental insults by exposure to the environment for a period of 1to 7 days. DNA was isolated and quantified as per the methods of Example4.

Example 11

Genomic DNA was isolated as described in Example 4, wherein thebiological samples were of varying nature, comprising buccal swabs onthe materials cotton, rayon, and nylon; blood, saliva and semen stainson materials like cotton cloth, denim, polyester cloth, FTA paper,filter paper; cigarette buts, swab of finger prints; mixtures of bodyfluids like epithelial cells and semen; swabs of body fluids ondifferent surfaces.

Example 12

Genomic DNA was isolated from biological samples, as described inExample 11, and were processed for quantification of human DNA using theQuantifiler® Human DNA Quantification Kit. The results for the quantityof human DNA and the C_(t) of the IPC, which measures the presence ofPCR inhibitors from some typical substrates containing biologicalmaterials, are presented in Table 8. A positive difference greater than1 in the IPC C_(t) value for a sample DNA preparation relative to theIPC C_(t) value of the negative template control (NTC) indicates thepresence of PCR inhibitors.

TABLE 8 Sample DNA Yield, ng IPC C_(t) Blood 2 μl  62 ± 13 27.6 BloodStain 2 μl on denim  76 ± 15 27.9 Blood Stain 2 μl on rayon 39.5 ± 8 27.7 Blood Stain 2 μl on nylon  39 ± 10 27.6 Saliva 5 μl 158 ± 30 27.6Saliva stain 5 μl on cotton 143 ± 25 27.7 Semen 2 μl 1250 ± 150 27.6Semen stain 2 μl on cotton 1340 ± 130 27.7 Cigarette butt 154 ± 40 27.8Chewing Gum 21 ± 8 27.9 Extraction Blank 0 27.6 NTC 27.6

Example 13

Genomic DNA was isolated from biological samples as described in Example11, and were processed for genotyping using an AmpFISTR® kit, such asIdentifiler®. The genotype profiles thus obtained from some typicalsubstrates containing biological materials are presented in FIG. 4.

Example 14

Genomic DNA was isolated from biological samples as described inExamples 1 to 12, and was processed using the reagents as describedtherein in the form of a kit.

What is claimed is:
 1. A kit for isolating and purifying nucleic acidfrom a sample comprising biological material, comprising a startingsolution comprising a soluble polymer and detergent; a polar solventcomprising ethanol; and dextran-encased magnetically attractableparticles.
 2. The kit of claim 1, wherein the polymer comprises one ormore of dextran, cellulose, cellulose derivatives, soluble starch,dextrin, cellodextrin, polyethylene glycol, heparin, glycogen, andcombinations thereof.
 3. The kit of claim 1, wherein the detergentcomprises one or more of N-lauroyl sarcosine, sarcosyl, deoxycholate,CTAB, deoxycholate, dodecyl β-D-maltoside, nonanoyl-N-methylglucamide,polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether, sodiumdodecyl sulfate, and combinations thereof.
 4. The kit of claim 1,wherein all of the contents of the kit are in one container.
 5. The kitof claim 1, further comprising a lysis solution.
 6. The kit of claim 1,further comprising a wash solution.